Materials Included In Kit

Additional Materials Required

Prelab Preparation

• Amylase solution, 1% (for Part A): Add 1 g of amylase to 100 mL of distilled or deionized water. Mix gently with a stirring rod to dissolve. Avoid vigorous mixing on a magnetic stirrer, which will cause excessive frothing or foaming and may denature the enzyme. For best results, prepare this solution fresh on the day of lab.
• Buffer solutions, pH 2, 7, 9 and 11 (for Part B): Prepare fresh using appropriate buffer capsules or buffer envelopes. Each capsule makes 100 mL of buffer solution.
• Glucose solution, 1%: Dissolve 1 g of glucose (dextrose) in 100 mL of distilled or deionized water. This solution is susceptible to mold formation and should be prepared within one week of use.
• Litmus–milk solution (for Part A): Add 100 mL of distilled or deionized water to 1 g of the litmus-milk powder to give a 1% solution. Prepare the solution within 2−3 days of use.
• Pepsin solution (acidified, for Part A): Prepare 100 mL of 0.01 M hydrochloric acid solution by appropriate dilution of a more concentrated solution (for example, by diluting 1 mL of 1 M HCl to 100 mL with water). Prepare the enzyme solution fresh the day of lab by dissolving 1 g of pepsin in 100 mL of 0.01 M HCl.
• Protein (albumin) solution, 2%: Add 10 mL of distilled or deionized water to 2 g of egg albumun (ovalbumin) and allow to soak for several hours in a bottle or flask to aid in dissolving without agitation or frothing. Add 90 mL of distilled or deionized water and gently shake the bottle or flask. For best results, prepare within one week of use and store in a refrigerator. Protein solutions are prone to molding.
• Starch solution, 1%: Add 1 g of starch to 100 mL of distilled or deionized water. Mix gently with stirring rod to dissolve.

Safety Precautions

Biuret test solution contains copper(II) sulfate in a concentrated sodium hydroxide solution. It is a corrosive liquid and will cause severe skin burns and serious eye damage. It is toxic by ingestion and harmful if swallowed. Iodine−potassium iodide solution causes skin and eye irritation and may be harmful if inhaled. The pepsin solution contains dilute hydrochloric acid and may also be irritating to the skin and eyes. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information. Remind students to wash hands thoroughly with soap and water before leaving the lab.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The reaction and product mixtures in Parts A and B may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. The acidic pepsin solution (Part A), and pH 2 buffer should be neutralized prior to disposal according to Flinn Suggested Disposal Method #24b. Remaining buffer solutions, as well as enzyme and substrate solutions, may also be rinsed down the drain with excess water. Save Benedict’s reagent, biuret solution and iodine for future use.

Lab Hints

• The laboratory work for this activity may be completed in a typical 2-hour lab period by dividing the pH optimization studies for amylase and pepsin in Part B among two pairs of students in a working group of four. Students may work in pairs on Part A to become familiar with the chemical tests and the observations of positive test results for all of the enzymes. The Prelaboratory Assignment should be assigned as preparation for lab and reviewed with students to ensure their familiarity with the enzymes and substrates for each reaction and the concept of optimum pH.
• The amylase−starch−iodine and lipase−buttermilk−litmus procedures are convenient for determining enzyme activity because the indicators (iodine and litmus, respectively) are “spectator reagents” that do not interfere with the enzyme reactions. The indicators’ changing colors allow students to measure the difference in the amount of time required for each reaction. The pepsin−albumin test reactions must proceed for a certain period of time before the biuret test solution is added because the basic biuret solution stops the reaction.
• The effect of pH on the rate of reaction of lipase also cannot be studied using the chemical test used in Part A because the pH buffer will affect the color of litmus. Litmus is an acid–base indicator that is pink or red in acidic solutions and blue or purple in base (including the basic buttermilk solution). Hydrolysis of milk fat by lipase releases fatty acids, which turn the indicator pink. Another convenient indicator that may be used to study lipase is phenol red.
• Pepsin and amylase are both very sensitive to pH making them ideal candidates for study in Part B. Incorporating trypsin in Part B would give an interesting comparison or contrast for the actions of different proteases. Digestion of proteins by trypsin occurs in the small intestine and its optimum pH is 9−11 rather than 2, as for pepsin. The biuret test can be used to study protein hydrolysis using both pepsin and trypsin. Recall that the biuret test solution is blue (due to the presence of copper ions) and turns purple when mixed with large polypeptides or proteins due to chelate/complex ion formation with at least two peptide linkages. Smaller peptides and amino acids form pink complexes with the biuret test solution.
• Many instructors like to include temperature optimization studies alongside pH profiles in enzyme activity labs. We have found that the optimum temperatures for the common digestive enzymes are significantly higher than physiological temperature and do not exhibit the expected bell-shaped curves. Results at different temperatures correlate more with the general effect of temperature on reaction rates rather than with the effect of temperature on protein stability and/or denaturation. Thus, all of the enzyme reactions studied in this experiment are very slow or nonexistent at 0–5 °C, but then also tend to accelerate in a regular manner up to and including 85–95 °C.

Answers to Prelab Questions

1. Complete the following concept map to summarize the digestion of carbohydrates, proteins and lipids.
   {14045_Answers_Figure_3}
2. Enzymes have an optimum pH at which they are most effective. The effect of pH on enzyme activity is due to interactions of acids and bases with amino acid side chains in the enzyme−protein structure. Use the model of enzyme action to explain how ionization of acidic or basic groups in a protein may influence substrate binding. Many amino acids have acidic (–CO₂^–) or basic (–NH₃⁺) groups in their side chains that will be charged or ionized. Ionization depends on pH. The charged side chains form ionic salt bridges that help determine the overall, three-dimensional “native” structure of the protein or enzyme. Changing the pH will alter the formation of these ionic bonds and may destabilize or distort the active site so that it does not bind, or binds less strongly, to the substrate. Weakening the enzyme–substrate binding constant will decrease the rate of the enzyme-catalyzed reaction.
3. Consult biochemistry or physiology textbooks or the Internet to research the properties of digestive enzymes. Fill in the blanks in the table below to show the substrate for each enzyme, the products of digestion and where digestion takes place. Estimate the optimal pH for each enzyme based upon where in the body digestion occurs.
   {14045_Answers_Table_2}

Sample Data

Part A. Chemical Tests for Enzyme Activity

Answers to Questions

Part A. Chemical Tests for Enzyme Activity

1. Compare and contrast the biuret test results in test tubes 1 and 2. Describe the evidence, if any, for the digestion of albumin using pepsin.

   Biuret gave a positive test for protein in test tube 1—a purple solution. No hydrolysis occurred. Addition of enzyme to test tube 2 gave a negative result for protein with biuret. The pink color is due to small peptides and amino acids produced by the hydrolysis of albumin.

2. What evidence was obtained for the breakdown of fat in the litmus−milk solution due to the action of lipase?

   The initial color of the litmus–milk solution was blue (neutral or basic). Breakdown of fat molecules by lipase was shown by the color change to pink, the acidic color of litmus, due to the formation of fatty acids.

3. Compare and contrast the iodine test results in test tubes 5 and 6. Describe the evidence for the digestion of starch using amylase.

   The starch–iodine mixture was dark blue. When amylase was present (test tube 6), the mixture turned yellow due to the digestion (hydrolysis) of starch molecules. Iodine is yellow.

4. Explain the Benedict’s test result observed for the products of the starch−amylase reaction in test tube 7.

   Digestion of starch by amylase produces maltose and glucose. Both of these are reducing sugars and should give a positive test with Benedict’s reagent. Test tube 7 turned green, a sign of a positive test result, although there was not as much reducing sugar as in test tube 8.

1. Compare the optimum pH results obtained for amylase and pepsin with those predicted in the Prelaboratory Assignment. Discuss any possible discrepancies between the predicted and experimental results.

   The optimum pH was 2 for the pepsin and 9–11 for amylase. The results for pepsin agree with predictions, since pepsin works in the stomach, which has an acidic pH. The results for amylase were different than predicted. It seems that the amylase may not be salivary amylase, but rather pancreatic amylase, which functions at a higher pH.

2. What was the purpose of including control samples containing only substrate and buffer (test tubes 1−4 for each enzyme) in this study? Did any of these mixtures show any reaction?

   In order to determine whether pH affects the rate of reaction with enzyme, we need to verify that changing just the pH does not cause hydrolysis or breakdown of the substrate molecules. The control samples gave negative results.

3. Protein digestion involves the hydrolysis of peptide linkages and is thus the reverse of protein synthesis, in which amino acids combine. Define the term hydrolysis and complete the following equation for the hydrolysis of a dipeptide.
   {14045_Answers_Equation_1}

   Hydrolysis is defined as the chemical decomposition or breakdown of a compound into smaller compounds by reaction with water.

4. The protease enzymes pepsin, trypsin and chymotrypsin are released in inactive forms called zymogens that must be cleaved and activated by hydrolysis prior to their role in protein digestion. How does this cleavage step help to regulate enzyme activity?

   Hydrolysis of the inactive forms of these enyzmes does not require cellular energy and only occurs when stimulated by the release of gastric juices (in the case of pepsin) or pancreatic juices (trypsin). The purpose of protease regulation is to prevent these enzymes from breaking down proteins in the body prior to being needed for digestion.

5. Manufacturers of nutritional supplements claim that ingesting powdered enzymes will increase the ability of the body to digest food. Critique (defend or criticize) this marketing position.

   There are a handful of cases where ingesting enzyme supplements is beneficial—in the case of known enzyme deficiency diseases, such as lactose intolerance or pancreatic insufficiency. In other cases, the body naturally produces the enzymes required for digestion. The efficiency of powdered enzymes is highly questionable since the body will probably break down the enzyme protein molecules before they can help you digest anything else.

Introduction

Organisms that do not make their own food must break down large macromolecules to generate the “building blocks of life.” Investigate the properties of digestive enzymes that break down plant and animal tissue—food—into glucose, amino acids and fatty acids needed for metabolism and growth.

Concepts

• Digestion
• Catalysts
• Enzymes
• Lock and key theory
• Protein structure
• pH

Background

The amazing transformation of food into simpler molecules is called digestion. Digestion occurs in the gastrointestinal (GI) tract or alimentary canal, a mucous–membrane lined tube that extends from the mouth to the large intestine. While in the GI tract, food is first mechanically broken down and then chemically treated with acids, bases and special enzymes within the organs of the digestive system.

Enzymes are biochemical catalysts. A catalyst is any substance that speeds up the rate of a chemical reaction without being permanently altered or consumed in the process. Almost all the chemical reactions that take place in living organisms are catalyzed by enzymes, which provide an alternative reaction pathway and lower activation energy, to accelerate the transformation of reactants into products. Enzymes are highly active catalysts, performing the same reaction thousands of times a second.

The function of an enzyme depends on its three-dimensional protein structure. Most enzymes are globular proteins with unique, characteristic shapes produced by a combination of diverse intramolecular attractive forces, including hydrogen bonding between peptide linkages as well as ionic bonding, hydrogen bonding and disulfide bonds between amino acid side chains. One region or section of the protein structure generally contains what is called the active site, which has a suitable shape, orientation and functionality to bind to the substrate(s) for a particular reaction. Because of the exclusive nature of enzyme−substrate binding, the human body contains thousands of different enzymes needed to catalyze all the different biochemical reactions that must occur.

The unique, functional shape of an enzyme with its active site is often compared to a lock into which the substrate will bind or fit like a key. This simple lock-and-key theory of enzyme structure and function was first proposed by Emil Fischer in 1894 (see Figure 1). Each type of substrate has a different shape and functional group reaction requiring a specific enzyme.

In 1958, Daniel Koshland modified the lock-and-key model to account for the actions of drugs and other small molecules that inhibit enzyme activity. According to Koshland’s induced-fit theory of enzyme action, the active site is not a perfect, preformed site for substrate binding. Binding by a substrate or inhibitor molecule changes the conformation of the protein in the active site region to match the size, shape and polarity of the molecule and enhance the binding constant. Non-covalent interactions between the enzyme and substrate stabilize it in a configuration that weakens bonds in the substrate and lowers the activation energy required to break those bonds. This feature accounts for the large rate acceleration provided by enzymatic catalysis.

Digestion begins in the mouth, where saliva provides the first chemical treatment of food. Saliva is composed of a neutral pH mixture of water, minerals, proteins, and the enzyme amylase. Amylase breaks down starch, a polysaccharide, into simpler carbohydrate molecules, principally, maltose and glucose. Glucose is the sugar used during cellular respiration as a source of energy. Digestion continues in the stomach with the release of hydrochloric acid and pepsinogen. Hydrochloric acid acts to denature or uncoil protein molecules in food and also activates pepsinogen, the inactive form of the enzyme pepsin. Pepsin is a protease that catalyzes the breakdown or hydrolysis of food proteins to smaller polypeptides and amino acids. Foods that are predominantly carbohydrates pass through the stomach quickly, followed by high-protein foods, and finally high-fat foods. Glucose, minerals and small amounts of water are absorbed through the walls of the stomach directly into the bloodstream for transport to the liver or to other cells in the body. Movements by the muscular stomach wall propel partially digested food from the stomach into the small intestine, where excretions from the pancreas, liver and small intestine combine to complete the digestive process.

The digestion of carbohydrates into glucose and other monosaccharides is completed in the small intestine by the enzymes sucrase, maltase, lactase and pancreatic amylase. Pancreatic juices also contain two additional proteases, trypsin and chymotrypsin as well as lipase, which hydrolyzes fats and other lipids into fatty acids. Bile salts aid the digestion of fats by acting like soap molecules, breaking down insoluble fat globules into smaller droplets and increasing the surface area for the action of pancreatic lipase.

Experiment Overview

The purpose of this experiment is to verify the actions of amylase, pepsin and lipase on proteins, starch and fat, respectively, using a series of chemical tests. These chemical tests will then be used to investigate the optimum pH conditions for amylase and pepsin. Students may work collaboratively in groups of four to complete the second part of the experiment, with one pair in each group studying amylase, the other pair studying pepsin.

Materials

Prelab Questions

1. Complete the following concept map to summarize the digestion of carbohydrates, proteins and lipids.
   {14045_PreLab_Figure_2}
2. Enzymes have an optimum pH at which they are most effective. The effect of pH on enzyme activity is due to interactions of acids and bases with amino acid side chains in the enzyme−protein structure. Use the model of enzyme action to explain how ionization of acidic or basic groups in a protein may influence substrate binding.
3. Consult biochemistry or physiology textbooks or the Internet to research the properties of digestive enzymes. Fill in the blanks in the table below to show the substrate for each enzyme, the products of digestion, and where digestion takes place. Estimate the optimal pH for each enzyme based upon where in the body digestion occurs.
   {14045_PreLab_Table_1}

Safety Precautions

Biuret test solution contains copper(II) sulfate in a concentrated sodium hydroxide solution. It is a corrosive liquid and will cause severe skin burns and serious eye damage. It is toxic by ingestion and harmful if swallowed. Iodine–potassium iodide solution causes skin and eye irritation and may be harmful if inhaled. The pepsin solution contains dilute hydrochloric acid and may also be irritating to the skin and eyes. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a lab coat or chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the lab.

Procedure

Part A. Chemical Tests for Enzyme Activity
Use a clean, graduated pipet or a designated eyedropper for each new substrate and enzyme solution (a total of eight disposable pipets are needed).

Pepsin

1. Label two test tubes 1 and 2. Using a clean, graduated pipet, add 3 mL of 2% protein solution (albumin) to test tube 1 and 1 mL of the albumin solution to test tube 2.
2. Add 2 mL of 1% pepsin to test tube 2.
3. Place both test tubes in a 45–50 °C water bath for five minutes.
4. Remove the test tubes from the water bath and, using a clean, graduated pipet, add 1 mL of biuret test solution to each test tube.
5. Observe and record the color and appearance of the samples in test tubes 1 and 2.

Lipase

6. Label two test tubes 3 and 4. Using a clean, graduated pipet, add 2 mL of the 1% litmus−milk solution to test tube 3 and 1 mL of the milk solution to test tube 4. Note: The litmus−milk solution contains buttermilk, a fat.
7. Add 1 mL of 1% lipase solution to test tube 4. Gently swirl the test tube to mix the contents. After three minutes, record the color and appearance of the mixtures in test tubes 3 and 4.

Amylase

8. Label four test tubes 5−8. Using a clean, graduated pipet, add 1 mL of 1% starch solution to each test tube 5, 6 and 7 (not 8).
9. Add 1 mL of 1% amylase solution to test tubes 6 and 7 only.
10. Add 1 mL of 1% glucose solution to test tube 8.
11. Gently swirl each test tube to mix the contents. Then place the test tubes in a beaker of hot tap water for two minutes.
12. Test the samples in test tubes 5 and 6 only for residual starch: Add 4−5 drops of iodine solution to each test tube. Record the color and appearance of each mixture.
13. Test the second starch−amylase mixture for the presence of glucose: Add 1 mL of Benedict’s reagent to each test tube 7 and 8. Note: Test tube 8 is a control or reference test to illustrate a positive test result for a reducing sugar.
14. Place test tubes 7 and 8 in a boiling water bath for 2−3 minutes. Observe the color and appearance of each mixture.
15. Discard the contents of test tubes 1−8 as directed by the instructor. Wash and rinse the test tubes with distilled or deionized water.

Part B. Effect of pH on Enzyme Activity
Each pair of students should study one enzyme, either amylase or pepsin. Note the control samples (test tubes 1–4) in each investigation. These samples do not contain enzyme. Carefully label all test tubes!

Amylase

1. Prepare 10 mL of 1% amylase in four different buffer solutions (pH 2, 7, 9 and 11) for use in steps 5–8. For each solution, dissolve 0.100 g of amylase in 10.0 mL of the appropriate buffer. Accurately measure both the enzyme mass and the liquid volume so that the enzyme concentration is constant in each solution. Use a graduated cylinder to transfer the buffer solution. Enzyme concentration is NOT a variable in these experiments!
2. Label eight test tubes and add 1 mL of 1% starch solution plus 2−3 drops of iodine−potassium iodide solution to each test tube 1−8.
3. To four of the test tubes (1−4), add 1 mL of the appropriate buffer solution as follows: Add pH 2 buffer to test tube 1, pH 7 buffer to test tube 2, pH 9 buffer to test tube 3 and pH 11 buffer to test tube 4. Place these test tubes in a beaker containing warm tap water (30–35 °C).
4. Start timing and observe if any of the control solutions decolorize within 5 minutes.
5. Stagger the starts for the different amylase buffer solutions in steps 5–8! Add 1 mL of the pH 2 buffer/amylase solution to test tube 5, place the test tube in a beaker with warm water (30–35 °C) and start timing. Record the time in seconds until the blue color disappears. If the tube does not decolorize in 5 minutes, write no reaction.
6. Add 1 mL of the pH 7 buffer/amylase solution to test tube 6, place the test tube in the warm water bath and start timing. Record the time in seconds until the blue color disappears. If the tube does not decolorize in 5 minutes, write no reaction.
7. Add 1 mL of the pH 9 buffer/amylase solution to test tube 7, place the test tube in the warm water bath and start timing. Record the time in seconds until the blue color disappears. If the tube does not decolorize in 5 minutes, write no reaction.
8. Add 1 mL of the pH 11 buffer/amylase solution to test tube 8, place the test tube in the warm water bath and start timing. Record the time in seconds until the blue color disappears. If the tube does not decolorize in 5 minutes, write no reaction.
9. Discard the contents of test tubes 1−8 as directed by the instructor. Wash and rinse the test tubes with distilled or deionized water.

Pepsin

10. Prepare a 45–50 °C water bath for use in step 15.
11. Prepare 10 mL of 1% pepsin in four different buffer solutions (pH 2, 7, 9 and 11) for use in step 14. For each solution, dissolve 0.100 g of pepsin in 10.0 mL of the appropriate buffer. Accurately measure both the enzyme mass and the liquid volume so that the enzyme concentration is constant in each solution. Use a graduated cylinder to transfer the buffer solution. Enzyme concentration is NOT a variable in these experiments!
12. Label eight test tubes. Add 1 mL of 1% albumin solution to each test tube 1−8.
13. To four of the test tubes (1−4), add 2 mL of the appropriate buffer solution as follows: Add pH 2 buffer to test tube 1, pH 7 buffer to test tube 2, pH 9 buffer to test tube 3 and pH 11 buffer to test tube 4.
14. To the next four test tubes (5−8), add 2 mL of the appropriate pepsin/buffer solution as follows: Add the pH 2 pepsin solution to test tube 5, the pH 7 solution to test tube 6, the pH 9 solution to test tube 7 and the pH 11 solution to test tube 8.
15. Place all eight test tubes in a 45–50 °C water bath for five minutes.
16. Remove the test tubes from the water bath and add 1 mL of biuret test solution to each test tube.
17. Record the color and appearance of the sample in each test tube.
18. Discard the contents of test tubes 1−8 as directed by the instructor. Wash and rinse the test tubes with distilled or deionized water.

Student Worksheet PDF

14045_Student1.pdf